Prevention of vascular calcification is mediated in part by Matrix Gla Protein (MGP) and the hepatic ABC transporter C6 (AbccG). MGP is a secreted matrix protein and may affect vascular calcification in vivo by binding bone morphogenetic proteins (BMP) and/or calcium through gamma-carboxylated glutamates. Our data show that in vascular cells BMP-2/4 induce expression of the activin-like kinase receptor 1 (ALK1), an essential TGF-3 receptor in vascular development. ALK1 is also expressed in atherosclerotic lesions and promotes aggregation and proliferation of cultured lesion cells. Induction of the ALK1 receptor allows BMP- 2/4 to regulate expression of vascular endothelial growth factor and MGP, which provides a feed back loop to limit BMP-activity. The importance of this pathway in atherogenesis is unknown. Abcc6 is a membrane transporter that is mainly expressed in the liver. AbccG was identified in our previous studies using an integrative genomics approach, and was shown to protect against vascular calcification. Abcc6 deficiency has been associated with pseudoxanthoma elasticum that is characterized by progressive calcification and accelerated atherosclerosis. The site and the mechanism of action of Abcc6 are not understood. This application focuses on the mechanisms by which MGP and Abcc6 prevent vascular calcification, and the effect of ALK1 and Abcc6 on atherogenesis. Four specific aims will be addressed. In Specific Aim 1; we will determine the importance of BMP-binding versus calcium-binding for the ability of MGP to inhibit vascular calcification in vivo using homologous recombination in mice. In Specific Aim 2, we will identify the receptor(s) and signaling pathway(s) that are required for BMP-2/4 to induce expression of the ALK1- receptor using siRNA, dominant negative receptors and molecular inhibitors in vitro. Specific Aim 3 will address Abcc6 and Abcc6 related pathways and determine the site and the molecular basis of its inhibitory effect on vascular calcification using both in vitro and in vivo techniques. Finally, in Specific Aim 4, we will address the effects of modulating expression of ALK1 and Abcc6 on lesion development in established mouse models of atherosclerosis.